High-level ceftazidime/avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated ?-lactamase CMY-185 variant

Objectives: To characterize a blaCMY variant associated with ceftazidime/avibactam resistance from a serially collected Escherichia coli isolate. Methods: A patient with an intra-abdominal infection due to recurrent E. coli was treated with ceftazidime/avibactam. On Day 48 of ceftazidime/avibactam therapy, E. coli with a ceftazidime/avibactam MIC of >256 mg/L was identified from abdominal drainage. Illumina and Oxford Nanopore Technologies WGS was performed on serial isolates to identify potential resistance mechanisms. Site-directed mutants of CMY ss-lactamase were constructed to identify amino acid residues responsible for ceftazidime/avibactam resistance. Results: WGS revealed that all three isolates were E. coli ST410. The ceftazidime/avibactam-resistant strain uniquely acquired a novel CMY beta-lactamase gene, herein called blaCMY-185, harboured on an IncI-./K1 conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2, including A114E, Q120K, V211S and N346Y, and conferred high-level ceftazidime/avibactam resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced ceftazidime/avibactam susceptibility. However, double and triple mutants containing N346Y previously associated with ceftazidime/avibactam resistance in other AmpC enzymes, conferred ceftazidime/avibactam MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to steric hindrance between the side chain of Y346 and the sulphate group of avibactam. Conclusions: We identified ceftazidime/avibactam resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer ceftazidime/avibactam resistance.